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Gamma-tubulin coordinates nuclear envelope assembly around chromatin

View Article: PubMed Central - PubMed

ABSTRACT

The cytosolic role of γ-tubulin as a microtubule organizer has been studied thoroughly, but its nuclear function is poorly understood. Here, we show that γ-tubulin is located throughout the chromatin of demembranated Xenopus laevis sperm and, as the nucleus is formed, γ-tubulin recruits lamin B3 and nuclear membranes. Immunodepletion of γ-tubulin impairs X. laevis assembly of both the lamina and the nuclear membrane. During nuclear formation in mammalian cell lines, γ-tubulin establishes a cellular protein boundary around chromatin that coordinates nuclear assembly of the daughter nuclei. Furthermore, expression of a γ-tubulin mutant that lacks the DNA-binding domain forms chromatin-empty nuclear like structures and demonstrate that a constant interplay between the chromatin-associated and the cytosolic pools of γ-tubulin is required and, when the balance between pools is impaired, aberrant nuclei are formed. We therefore propose that the nuclear protein meshwork formed by γ-tubulin around chromatin coordinates nuclear formation in eukaryotic cells.

No MeSH data available.


Related in: MedlinePlus

γ-Tubulin promotes the formation of the nuclear envelope in the absence of lamin B3. (A) His6–lamin B3 (laminB3) was added back to Ksperm (Cont.; Ksperm without laminB3) and γ-tubulin immunodepleted egg extract (Depl.) and nuclear assembly was performed as in Fig. 5. Graph shows the mean percentage of formed nuclei in stage 3 and 4 in immunodepleted egg extracts and sperm (Depl. γTub) versus a control (black bar; Depl. Cont.), with addition of His6–lamin B3 (grey bar) to the γ-tubulin depleted extracts (± s.d., n = 3; * p < 0.05), as indicated. The western blot shows the amount of His6–lamin B3 added to sperm. To relate the amount of His6–lamin B3 to the amount present in extracts, 1 μl and 5 μl of egg extracts were loaded. Representative confocal fluorescence images of morphological changes of nuclei show the location of endogenous γ-tubulin (eγTub, green) or lamin B (red). (B) Representative confocal fluorescence images of morphological changes of nuclei in nuclear assembly reactions that were triggered by addition of lamin B3 immunodepleted egg extracts to sperm and incubated 90 min before fixation. The protein levels of lamin B3 in extracts were analyzed by WB. The graph shows the mean percentage of formed nuclei with γ-tubulin localized throughout the nuclei (black bar) or marginalized to the nuclear envelope (open bar) (± s.d., n = 3). (A, B) Localization of γ-tubulin (eγTub; green), His6–lamin B3 (lamin B; red) and lamin B3 (lamin B; red) were examined by immunofluorescence staining with the indicated antibody and nuclear membranes and nuclei were detected with Nile red (red) and DAPI (blue), respectively. The figure shows representative images from at least five experiments. Scale bars, 10 μm.
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fig0030: γ-Tubulin promotes the formation of the nuclear envelope in the absence of lamin B3. (A) His6–lamin B3 (laminB3) was added back to Ksperm (Cont.; Ksperm without laminB3) and γ-tubulin immunodepleted egg extract (Depl.) and nuclear assembly was performed as in Fig. 5. Graph shows the mean percentage of formed nuclei in stage 3 and 4 in immunodepleted egg extracts and sperm (Depl. γTub) versus a control (black bar; Depl. Cont.), with addition of His6–lamin B3 (grey bar) to the γ-tubulin depleted extracts (± s.d., n = 3; * p < 0.05), as indicated. The western blot shows the amount of His6–lamin B3 added to sperm. To relate the amount of His6–lamin B3 to the amount present in extracts, 1 μl and 5 μl of egg extracts were loaded. Representative confocal fluorescence images of morphological changes of nuclei show the location of endogenous γ-tubulin (eγTub, green) or lamin B (red). (B) Representative confocal fluorescence images of morphological changes of nuclei in nuclear assembly reactions that were triggered by addition of lamin B3 immunodepleted egg extracts to sperm and incubated 90 min before fixation. The protein levels of lamin B3 in extracts were analyzed by WB. The graph shows the mean percentage of formed nuclei with γ-tubulin localized throughout the nuclei (black bar) or marginalized to the nuclear envelope (open bar) (± s.d., n = 3). (A, B) Localization of γ-tubulin (eγTub; green), His6–lamin B3 (lamin B; red) and lamin B3 (lamin B; red) were examined by immunofluorescence staining with the indicated antibody and nuclear membranes and nuclei were detected with Nile red (red) and DAPI (blue), respectively. The figure shows representative images from at least five experiments. Scale bars, 10 μm.

Mentions: Interference with the function of lamin B3 affects the size of the formed nuclei (Lourim et al., 1996). To test whether the defects in nuclear assembly observed following γ-tubulin depletion are due to an impaired lamina formation or lamin B3 recruitment to chromatin, we added recombinant lamin B3 to Ksperm (Fig. 6A). In the absence of chromatin-bound γ-tubulin, lamin B3 did not associate with Ksperm and, consequently, addition of recombinant lamin B3 was not sufficient to trigger neither lamina nor nuclear formation (Fig. 6A), suggesting that lamin B3 needs to be recruited to chromatin by γ-tubulin.


Gamma-tubulin coordinates nuclear envelope assembly around chromatin
γ-Tubulin promotes the formation of the nuclear envelope in the absence of lamin B3. (A) His6–lamin B3 (laminB3) was added back to Ksperm (Cont.; Ksperm without laminB3) and γ-tubulin immunodepleted egg extract (Depl.) and nuclear assembly was performed as in Fig. 5. Graph shows the mean percentage of formed nuclei in stage 3 and 4 in immunodepleted egg extracts and sperm (Depl. γTub) versus a control (black bar; Depl. Cont.), with addition of His6–lamin B3 (grey bar) to the γ-tubulin depleted extracts (± s.d., n = 3; * p < 0.05), as indicated. The western blot shows the amount of His6–lamin B3 added to sperm. To relate the amount of His6–lamin B3 to the amount present in extracts, 1 μl and 5 μl of egg extracts were loaded. Representative confocal fluorescence images of morphological changes of nuclei show the location of endogenous γ-tubulin (eγTub, green) or lamin B (red). (B) Representative confocal fluorescence images of morphological changes of nuclei in nuclear assembly reactions that were triggered by addition of lamin B3 immunodepleted egg extracts to sperm and incubated 90 min before fixation. The protein levels of lamin B3 in extracts were analyzed by WB. The graph shows the mean percentage of formed nuclei with γ-tubulin localized throughout the nuclei (black bar) or marginalized to the nuclear envelope (open bar) (± s.d., n = 3). (A, B) Localization of γ-tubulin (eγTub; green), His6–lamin B3 (lamin B; red) and lamin B3 (lamin B; red) were examined by immunofluorescence staining with the indicated antibody and nuclear membranes and nuclei were detected with Nile red (red) and DAPI (blue), respectively. The figure shows representative images from at least five experiments. Scale bars, 10 μm.
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Related In: Results  -  Collection

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fig0030: γ-Tubulin promotes the formation of the nuclear envelope in the absence of lamin B3. (A) His6–lamin B3 (laminB3) was added back to Ksperm (Cont.; Ksperm without laminB3) and γ-tubulin immunodepleted egg extract (Depl.) and nuclear assembly was performed as in Fig. 5. Graph shows the mean percentage of formed nuclei in stage 3 and 4 in immunodepleted egg extracts and sperm (Depl. γTub) versus a control (black bar; Depl. Cont.), with addition of His6–lamin B3 (grey bar) to the γ-tubulin depleted extracts (± s.d., n = 3; * p < 0.05), as indicated. The western blot shows the amount of His6–lamin B3 added to sperm. To relate the amount of His6–lamin B3 to the amount present in extracts, 1 μl and 5 μl of egg extracts were loaded. Representative confocal fluorescence images of morphological changes of nuclei show the location of endogenous γ-tubulin (eγTub, green) or lamin B (red). (B) Representative confocal fluorescence images of morphological changes of nuclei in nuclear assembly reactions that were triggered by addition of lamin B3 immunodepleted egg extracts to sperm and incubated 90 min before fixation. The protein levels of lamin B3 in extracts were analyzed by WB. The graph shows the mean percentage of formed nuclei with γ-tubulin localized throughout the nuclei (black bar) or marginalized to the nuclear envelope (open bar) (± s.d., n = 3). (A, B) Localization of γ-tubulin (eγTub; green), His6–lamin B3 (lamin B; red) and lamin B3 (lamin B; red) were examined by immunofluorescence staining with the indicated antibody and nuclear membranes and nuclei were detected with Nile red (red) and DAPI (blue), respectively. The figure shows representative images from at least five experiments. Scale bars, 10 μm.
Mentions: Interference with the function of lamin B3 affects the size of the formed nuclei (Lourim et al., 1996). To test whether the defects in nuclear assembly observed following γ-tubulin depletion are due to an impaired lamina formation or lamin B3 recruitment to chromatin, we added recombinant lamin B3 to Ksperm (Fig. 6A). In the absence of chromatin-bound γ-tubulin, lamin B3 did not associate with Ksperm and, consequently, addition of recombinant lamin B3 was not sufficient to trigger neither lamina nor nuclear formation (Fig. 6A), suggesting that lamin B3 needs to be recruited to chromatin by γ-tubulin.

View Article: PubMed Central - PubMed

ABSTRACT

The cytosolic role of &gamma;-tubulin as a microtubule organizer has been studied thoroughly, but its nuclear function is poorly understood. Here, we show that &gamma;-tubulin is located throughout the chromatin of demembranated Xenopus laevis sperm and, as the nucleus is formed, &gamma;-tubulin recruits lamin B3 and nuclear membranes. Immunodepletion of &gamma;-tubulin impairs X. laevis assembly of both the lamina and the nuclear membrane. During nuclear formation in mammalian cell lines, &gamma;-tubulin establishes a cellular protein boundary around chromatin that coordinates nuclear assembly of the daughter nuclei. Furthermore, expression of a &gamma;-tubulin mutant that lacks the DNA-binding domain forms chromatin-empty nuclear like structures and demonstrate that a constant interplay between the chromatin-associated and the cytosolic pools of &gamma;-tubulin is required and, when the balance between pools is impaired, aberrant nuclei are formed. We therefore propose that the nuclear protein meshwork formed by &gamma;-tubulin around chromatin coordinates nuclear formation in eukaryotic cells.

No MeSH data available.


Related in: MedlinePlus